Ranjan Chrisanthar

MDM2 plays a key role in modulating p53 function. The MDM2 SNP309T > G promoter polymorphism enhances Sp1 binding and has been linked to cancer risk and young age at diagnosis although with conflicting evidence. We report a second MDM2 promoter polymorphism, SNP285G > C, residing on the SNP309G allele. SNP285C occurs in Caucasians only, where 7.7% (95% CI 7.6%-7.8%) of healthy individuals carry the SNP285C/309G haplotype. In vitro analyses reveals that SNP309G enhances but SNP285C strongly reduces Sp1 promoter binding. Comparing MDM2 promoter status among different cohorts of ovarian (n = 1993) and breast (n = 1973) cancer patients versus healthy controls (n = 3646), SNP285C reduced the risk of both ovarian (OR 0.74; CI 0.58-0.94) and breast cancer (OR 0.79; CI 0.62-1.00) among SNP309G carriers.

Chemoresistance is the main obstacle to cure in most malignant diseases. Anthracyclines are among the main drugs used for breast cancer therapy and in many other malignant conditions. Single parameter analysis or global gene expression profiles have failed to identify mechanisms causing in vivo resistance to anthracyclines. While we previously found TP53 mutations in the L2/L3 domains to be associated with drug resistance, some tumors harboring wild-type TP53 were also therapy resistant. The aim of this study was; 1) To explore alterations in the TP53 gene with respect to resistance to a regular dose epirubicin regimen (90 mg/m(2) every 3 week) in patients with primary, locally advanced breast cancer; 2) Identify critical mechanisms activating p53 in response to DNA damage in breast cancer; 3) Evaluate in vitro function of Chk2 and p14 proteins corresponding to identified mutations in the CHEK2 and p14((ARF)) genes; and 4) Explore potential CHEK2 or p14((ARF)) germline mutations with respect to family cancer incidence.Snap-frozen biopsies from 109 patients collected prior to epirubicin (as preoperative therapy were investigated for TP53, CHEK2 and p14((ARF)) mutations by sequencing the coding region and p14((ARF)) promoter methylations. TP53 mutations were associated with chemoresistance, defined as progressive disease on therapy (p = 0.0358; p = 0.0136 for mutations affecting p53 loop domains L2/L3). Germline CHEK2 mutations (n = 3) were associated with therapy resistance (p = 0.0226). Combined, mutations affecting either CHEK2 or TP53 strongly predicted therapy resistance (p = 0.0101; TP53 mutations restricted to the L2/L3 domains: p = 0.0032). Two patients progressing on therapy harbored the CHEK2 mutation, Arg95Ter, completely abrogating Chk2 protein dimerization and kinase activity. One patient (Epi132) revealed family cancer occurrence resembling families harboring CHEK2 mutations in general, the other patient (epi203) was non-conclusive. No mutation or promoter hypermethylation in p14((ARF)) were detected.This study is the first reporting an association between CHEK2 mutations and therapy resistance in human cancers and to document mutations in two genes acting direct up/down-stream to each other to cause therapy failure, emphasizing the need to investigate functional cascades in future studies.

TP53 mutations have been associated with resistance to anthracyclines but not to taxanes in breast cancer patients. The MDM2 promoter single nucleotide polymorphism (SNP) T309G increases MDM2 activity and may reduce wild-type p53 protein activity. Here, we explored the predictive and prognostic value of TP53 and CHEK2 mutation status together with MDM2 SNP309 genotype in stage III breast cancer patients receiving paclitaxel or epirubicin monotherapy.Each patient was randomly assigned to treatment with epirubicin 90 mg/m(2) (n = 109) or paclitaxel 200 mg/m(2) (n = 114) every 3rd week as monotherapy for 4-6 cycles. Patients obtaining a suboptimal response on first-line treatment requiring further chemotherapy received the opposite regimen. Time from last patient inclusion to follow-up censoring was 69 months. Each patient had snap-frozen tumor tissue specimens collected prior to commencing chemotherapy.While TP53 and CHEK2 mutations predicted resistance to epirubicin, MDM2 status did not. Neither TP53/CHEK2 mutations nor MDM2 status was associated with paclitaxel response. Remarkably, TP53 mutations (p = 0.007) but also MDM2 309TG/GG genotype status (p = 0.012) were associated with a poor disease-specific survival among patients having paclitaxel but not patients having epirubicin first-line. The effect of MDM2 status was observed among individuals harbouring wild-type TP53 (p = 0.039) but not among individuals with TP53 mutated tumors (p>0.5).TP53 and CHEK2 mutations were associated with lack of response to epirubicin monotherapy. In contrast, TP53 mutations and MDM2 309G allele status conferred poor disease-specific survival among patients treated with primary paclitaxel but not epirubicin monotherapy.

Mutations affecting p53 or its upstream activator Chk2 are associated with resistance to DNA-damaging chemotherapy in breast cancer. ATM (Ataxia Telangiectasia Mutated protein) is the key activator of p53 and Chk2 in response to genotoxic stress. Here, we sought to evaluate ATM's potential role in resistance to chemotherapy.We sequenced ATM and assessed gene expression levels in pre-treatment biopsies from 71 locally advanced breast cancers treated in the neoadjuvant setting with doxorubicin monotherapy or mitomycin combined with 5-fluorouracil. Findings were confirmed in a separate patient cohort treated with epirubicin monotherapy. Each tumor was previously analyzed for CHEK2 and TP53 mutation status.While ATM mutations were not associated with chemo-resistance, low ATM expression levels predicted chemo-resistance among patients with tumors wild-type for TP53 and CHEK2 (P = 0.028). Analyzing the ATM-chk2-p53 cascade, low ATM levels (defined as the lower 5 to 50% percentiles) or mutations inactivating TP53 or CHEK2 robustly predicted anthracycline resistance (P-values varying between 0.001 and 0.027 depending on the percentile used to define "low" ATM levels). These results were confirmed in an independent cohort of 109 patients treated with epirubicin monotherapy. In contrast, ATM-levels were not suppressed in resistant tumors harboring TP53 or CHEK2 mutations (P > 0.5).Our data indicate loss of function of the ATM-Chk2-p53 cascade to be strongly associated with resistance to anthracycline/mitomycin-containing chemotherapy in breast cancer.

Expanding knowledge, together with implementation of new techniques, has fuelled the area of translational medical research aiming at improving prognostication as well as prediction in cancer therapy. At the same time, new discoveries have revealed a biological complexity we were unaware of only a decade ago. Thus, we are faced with novel challenges with respect to how we may explore issues such as prognostication and predict drug resistance in vivo. While microarray analysis exploring expression of thousands of genes in concert represents a major methodological advancement, discoveries such as the finding of different mechanisms of epigenetic silencing, intronic mutations, that most gene transcripts in the human genome are subject to alternative splicing and that hypersplicing seems to be a tumour-related phenomenon, exemplifies a complex pathology that may not be explored with use of single analytical methods only. This paper discusses clinical settings for studying drug resistance in vivo together with a discussion of contemporary biology in this field. Notably, each individual parameter which has been found correlated to drug resistance in vivo so far represents either a direct drug target or a factor involved in DNA repair or apoptosis. On the basis of these findings, we suggest drug resistance may be explored on the basis of upfront biological hypotheses.

Background: The role of normal tissue gene promoter methylation in cancer risk is poorly understood. Objective: To assess associations between normal tissue BRCA1 methylation and ovarian cancer risk. Design: 2 case–control (initial and validation) studies. Setting: 2 hospitals in Norway (patients) and a population-based study (control participants). Participants: 934 patients and 1698 control participants in the initial study; 607 patients and 1984 control participants in the validation study. Measurements: All patients had their blood sampled before chemotherapy. White blood cell (WBC) BRCA1 promoter methylation was determined by using methylation-specific quantitative polymerase chain reaction, and the percentage of methylation-positive samples was compared between population control participants and patients with ovarian cancer, including the subgroup with high-grade serous ovarian cancer (HGSOC). Results: In the initial study, BRCA1 methylation was more frequent in patients with ovarian cancer than control participants (6.4% vs. 4.2%; age-adjusted odds ratio [OR], 1.83 [95% CI, 1.27 to 2.63]). Elevated methylation, however, was restricted to patients with HGSOC (9.6%; OR, 2.91 [CI, 1.85 to 4.56]), in contrast to 5.1% and 4.0% of patients with nonserous and low-grade serous ovarian cancer (LGSOC), respectively. These findings were replicated in the validation study (methylation-positive status in 9.1% of patients with HGSOC vs. 4.3% of control participants—OR, 2.22 [CI 1.40 to 3.52]—4.1% of patients with nonserous ovarian cancer, and 2.7% of those with LGSOC). The results were not influenced by tumor burden, storage time, or WBC subfractions. In separate analyses of young women and newborns, BRCA1 methylation was detected in 4.1% (CI, 1.8% to 6.4%) and 7.0% (CI, 5.0% to 9.1%), respectively. Limitations: Patients with ovarian cancer were recruited at the time of diagnosis in a hospital setting. Conclusion: Constitutively normal tissue BRCA1 promoter methylation is positively associated with risk for HGSOC. Primary Funding Source: Norwegian Cancer Society.

Chemoresistance is the main obstacle to cancer cure. Contrasting studies focusing on single gene mutations, we hypothesize chemoresistance to be due to inactivation of key pathways affecting cellular mechanisms such as apoptosis, senescence, or DNA repair. In support of this hypothesis, we have previously shown inactivation of either TP53 or its key activators CHK2 and ATM to predict resistance to DNA damaging drugs in breast cancer better than TP53 mutations alone. Further, we hypothesized that redundant pathway(s) may compensate for loss of p53‐pathway signaling and that these are inactivated as well in resistant tumour cells. Here, we assessed genetic alterations of the retinoblastoma gene (RB1) and its key regulators: Cyclin D and E as well as their inhibitors p16 and p27. In an exploratory cohort of 69 patients selected from two prospective studies treated with either doxorubicin monotherapy or 5‐FU and mitomycin for locally advanced breast cancers, we found defects in the pRB‐pathway to be associated with therapy resistance (p‐values ranging from 0.001 to 0.094, depending on the cut‐off value applied to p27 expression levels). Although statistically weaker, we observed confirmatory associations in a validation cohort from another prospective study (n = 107 patients treated with neoadjuvant epirubicin monotherapy; p‐values ranging from 7.0 × 10−4 to 0.001 in the combined data sets). Importantly, inactivation of the p53‐and the pRB‐pathways in concert predicted resistance to therapy more strongly than each of the two pathways assessed individually (exploratory cohort: p‐values ranging from 3.9 × 10−6 to 7.5 × 10−3 depending on cut‐off values applied to ATM and p27 mRNA expression levels). Again, similar findings were confirmed in the validation cohort, with p‐values ranging from 6.0 × 10−7 to 6.5 × 10−5 in the combined data sets. Our findings strongly indicate that concomitant inactivation of the p53‐ and pRB‐ pathways predict resistance towards anthracyclines and mitomycin in breast cancer in vivo.

PTEN is an important tumor suppressor in breast cancer. Here, we examined the prognostic and predictive value of PTEN and PTEN pseudogene (PTENP1) gene expression in patients with locally advanced breast cancer given neoadjuvant chemotherapy.The association between pretreatment PTEN and PTENP1 gene expression, response to neoadjuvant chemotherapy, and recurrence-free and disease-specific survival was assessed in 364 patients with locally advanced breast cancer given doxorubicin, 5-fluorouracil/mitomycin, or epirubicin versus paclitaxel in three phase II prospective studies. Further, protein expression of PTEN or phosphorylated Akt, S6 kinase, and 4EBP1 was assessed in a subgroup of 187 tumors.Neither PTEN nor PTENP1 gene expression level predicted response to any of the chemotherapy regimens tested (n = 317). Among patients without distant metastases (n = 282), a high pretreatment PTEN mRNA level was associated with inferior relapse-free (RFS; p = 0.001) and disease-specific survival (DSS; p = 0.003). Notably, this association was limited to patients harboring TP53 wild-type tumors (RFS; p = 0.003, DSS; p = 0.009). PTEN mRNA correlated significantly with PTENP1 mRNA levels (r s = 0.456, p < 0.0001) and PTEN protein staining (r s = 0.163, p = 0.036). However, no correlation between PTEN, phosphorylated Akt, S6 kinase or 4EBP1 protein staining, and survival was recorded. Similarly, no correlation between PTENP1 gene expression and survival outcome was observed.High intratumoral PTEN gene expression was associated with poor prognosis in patients with locally advanced breast cancers harboring wild-type TP53.

p21 is a main effector of growth arrest induced by p53. In addition, a second transcript from the same gene (p21B) has been linked to apoptosis. We previously analyzed p21 status in breast cancer and reported two novel polymorphisms of the p21 gene. In the present study, we present a larger study designed to explore a possible association between these novel polymorphisms and breast cancer.The p21/p21B polymorphisms were analyzed in 507 breast cancer patients and 1,017 healthy individuals using cDNA or genomic DNA from tumor and/or blood samples.We detected five polymorphisms of the p21 gene. Three of these polymorphisms are earlier reported by others, whereas two were reported for the first time in a recent study by us. The presence of the A allele of the p21G251A polymorphism was observed more frequently among patients with primary stage III breast cancer (4.5%) compared with stage I and II tumors (1.5%) and healthy female controls (1.4%; P = 0.007, comparing the three groups; P = 0.0049 and P = 0.0057, comparing locally advanced to stage I/II and healthy controls, or to healthy controls alone, respectively). The allele frequencies of the remaining four polymorphisms were evenly distributed among patients and healthy individuals.The finding of an association between locally advanced breast cancer and one particular polymorphism of the p21 gene suggests this polymorphism to be related to tumor behavior, including enhanced growth rate. If confirmed in other studies, this may add significant information to our understanding of the biology as well as of the clinical behaviour of locally advanced breast cancers.

p21(WAF1/CIP1), transcribed from the CDKN1A locus, plays a key role executing p53-induced growth arrest. The recent discovery that an alternative transcript, p21B, induces apoptosis, suggests an additional important function of this gene. Here, we report p21 and p21B mutation status in large cohorts of breast cancers and compare distributions of p21B polymorphisms in cancer patients to healthy controls. In 521 breast tumor samples analyzed, only one point mutation affecting the p21B protein was observed. No mutations were found when screening a panel of 20 established cell lines. A novel polymorphism, p21B(G128T) was identified. Haplotype analysis revealed no association between this variant and the previously identified p21B polymorphism p21B(T35C) or any of the known p21(WAF1/CIP1) polymorphisms. As previously reported for p21B(T35C), distribution of p21B(G128T) was similar among breast cancer patients and healthy controls (n = 691 and 1,015; incidence 6.1 vs. 4.8%; p = 0.273, respectively). No association between p21B(G128T) or p21B(T35C) and response to chemotherapy with an anthracycline-containing regimen or paclitaxel was recorded. Our findings do not suggest mutations or polymorphisms of p21B to play a major role with respect to either breast cancer risk or sensitivity towards chemotherapy.

We read with great interest the recent article by Galmarini and colleagues in the journal.1 The authors reported a point mutation in exon 2 of the p21 gene (WAF1, CIP1, CDKN1A), leading to a premature STOP in codon 127 and paclitaxel resistance in noncancerous epithelial breast cells. The cyclin-dependent kinase inhibitor p21 is a main effector of growth arrest induced by p53. The role of p21 in breast cancer is still unclear, particularly with respect to drug resistance. Thus, if corroborated by clinical findings, identification of p21 as a key element for response to paclitaxel would be a major breakthrough to our understanding of drug resistance. Several groups have addressed the potential role of p21 polymorphisms as a risk factor for cancer development.2, 3, 4, 5 However, we are not aware of any study exploring p21 polymorphism or mutation status in relation to response to paclitaxel or other chemotherapy treatment in breast or other type of cancers in vivo. For several years, we have studied potential causes of resistance to chemotherapy in vivo focusing on genetic changes in TP53 and functionally associated genes.6, 7 As part of this work, we have studied genetic alterations (screening of the full coding sequence) and epigenetic changes (methylation analyses of the promoter) in p21. In a total of 521 breast cancer patients (320 stage III and 201 metastatic) studied in our reported and ongoing investigations, no mutations were observed in this material. Nor was hypermethylation of the p21 promoter region recorded.8 We identified 3 novel polymorphisms p21T234C, p21T330C and p21G528A in addition to the recently reported p21G251A which was found to be associated with locally advanced breast cancer,9 none of the polymorphisms were associated either with response to an anthracycline- or mitomycin-containing regimen (n = 214 patients) or to paclitaxel monotherapy (n = 106 patients). On the basis of these findings, we believe that disturbances in p21 function are unlikely to play a major role in paclitaxel resistance in breast cancer in vivo. However, the finding of Galmarini et al points to a role for the p21 pathway in cellular response to paclitaxel treatment, and it may well be that inactivation of other genes involved in the same pathway10 may be a mechanism causing paclitaxel resistance in human breast cancer. As such, the paper by Galmarini et al points to a potential important mechanism of drug resistance, advocating analysis of factors interacting with p21 function in different malignancies currently treated with paclitaxel. Yours sincerely Ranjan Chrisanthar* , Stian Knappskog* , Vidar Staalesen* , Johan Richard Lillehaug , Per Eystein Lønning*, * Institute of Medicine, Section of Oncology, Haukeland University Hospital, N-5021 Bergen, Norway, Department of Molecular Biology, University of Bergen, N-5020 Bergen, Norway.

Although gene expression microarrays provide novel tools and hold great promise in cancer research, achievements thus far in terms of improved prognostication and, in particular, prediction of drug sensitivity have been moderate. To improve clinical therapy, we believe that it is imperative to integrate gene expression arrays with other laboratory methods based on functional concepts [1,2].

5029 Background: Recently, some studies have reported BRCA1 WBC hypermethylation to be associated with increased risk of breast cancer. We assessed WBC BRCA1 promoter methylations in ovarian (OC) and breast cancer (BC) patients and healthy controls. In an OC subgroup harboring WBC BRCA1 methylation, we confirmed promoter methylation status in normal tissue. Methods: WBC DNA from 899 OC patients, 425 BC patients and 719 healthy controls were analyzed for BRCA1 promoter methylation by methylation specific PCR. In addition, we analyzed WBC DNA from 256 OC and 393 BC patients in blood samples drawn prior to diagnosis in a population-based study. We also analyzed 24 BC patients with a family history (BRCAPRO scores > 80%; Manchester score >40) without BRCA1/2 mutations. Paraffin-embedded normal tissue from 5 OC patients harboring WBC BRCA1 methylation was analyzed for BRCA1 methylation status. Finally, to determine potential risk factors in cis, we investigated BRCA1 haplotype status in a sub-cohort of 10 individuals with WBC methylated BRCA1and 13 controls. Results: We detected WBC BRCA1 promoter hypermethylation in 2.4% of healthy controls and 3.1% of BC patients (all BC individuals). No difference in BRCA1 methylation incidence was recorded between BC patients with blood samples drawn before (4.1%) or after (2.1%) diagnosis. In contrast, we detected WBC BRCA1 methylation in 10.3% of OC patients having blood samples drawn at diagnosis. Among OC patients with blood sampling 0-13 years (median 4.6 y) prior to diagnosis, BRCA1 promoter methylation was detected in 6.6%. BRCA1 promoter methylation was associated with a non-significant elevated risk of BC (HR 1.302; 95% CI 0.697-2.431) but a significantly increased risk of OC in the cohort of patients with blood samples drawn at time of (OR 4.765; CI 2.814-8.069) or prior to (OR 2.937; CI 1.476-5.845) diagnosis. We confirmed BRCA1 promoter methylation in normal tissue from all 5 individuals analyzed, excluding WBC promoter methylation being due to circulating DNA contamination. No association between BRCA1methylation and promoter haplotype was found. Conclusions: WBC BRCA1 promoter methylation is associated with increased risk of ovarian cancer.This finding has clinical as well as biological implications..

Abstract Background: Mutations in TP53 and its upstream p53-activator Chk2, are known to be associated with resistance to anthracycline and mitomycin treatment in locally advanced breast cancer. However, this association is not fully predictive, as some tumours are therapy resistant despite harbouring wild-type TP53 and CHK2. ATM is an upstream activator of both p53 and Chk2. Here, we explored ATM mutational status and expression levels with respect to anthracycline/mitomycine resistance in locally advanced breast cancers having primary chemotherapy with doxorubicin or 5FU-mitomycin and previously analyzed for TP53 and CHK2 status. Methods: In order to investigate ATM's potential predictive role, we performed MLPA-analysis, qPCR on ATM mRNA, ATM promoter methylation analyses and complete sequencing of the entire ATM coding region in patient biopsies from two prospective studies on resistance to doxorubicin and 5-FU/mitomycin in locally advance breast cancer (n=36 and n=38, respectively). Results: We performed complete sequencing of the ATM coding exons in 74 patients including 17 patients revealing primary resistance to chemotherapy. ATM mutations were detected in tumours from 5 patients. No mutation was detected among tumors resistant to chemotherapy. In addition, we observed 9 polymorphic variants present in more than one tumour, affecting or not affecting the amino acid sequence of the ATM protein (n = 34 patients in total). No correlation was observed between polymorphism status and response to therapy. No methylation of the ATM promoter, large deletions or duplication/amplifications of the ATM gene was observed. ATM expression levels varied substantially, with a ratio of 49 between the highest versus lowest level recorded. Among patients with no mutations in TP53 or CHK2, low ATM expression levels were significantly correlated to lack of response to doxorubicin or 5-FU/mitomycin (p=0.023; Mann-Whitney test). In contrast, ATM-levels were not suppressed in non-responding tumors harbouring TP53 or CHK2 mutations (p=0.642; Mann-Whitney test). Defining ATM mRNA levels in the lower 50%, 33% or 25% of the patient cohort to be reduced, tumors with either reduced levels of ATM or mutations affecting TP53 or CHK2 revealed a high risk for therapy resistance (p=0.004, p=0.001 and p=0.006, using the different ATM-level cut-offs; Fischer exact test). Interestingly, in a similar prospective study with a smaller number of patients displaying progressive disease upon treatment with epirubicin (n = 10), we find 2 out 5 TP53 and CHK2-wild-type non-responding tumors to express very low levels of ATM (within the lowest 5% of the cohort). Conclusions: Our data suggests that low expression of ATM may substitute for TP53 and CHK2 mutations causing resistance to anthracycline-and mitomycin therapy in breast cancer. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-01-02.